ProjectSummary/Abstract Classswitchrecombination(CSR)isageneticprocesswhereaBcellswitchesantibodyisotype productionthroughsite-specificintra-chromosomalDNArearrangementstimulatedbytheformation ofDNAdouble-strandbreaks(DSBs)attheimmunoglobulinheavychain(IgH)locus.DSBsare normallyrepairedbythenon-homologousend-joining(NHEJ)andalternative-endjoining(alt-EJ) DNArepairpathways.DuringCSR,DSBformationishighlyregulatedinvolvingacomplexinterplayof transcriptionalactivation,proteinrecruitmentandchromatinreorganization.Understandingthefactors regulatingDSBformationandrepairhasahighimpactonlymphomagenesis.Rloopsarethree strandedRNA:DNAhybridstructuresformedatIgHduringCSR.WhileRloopsareimplicatedin promotingDSBformationatIgH,theirroleinclassswitchrecombinationremainsundefined.Wefind thatmicedefectiveforRloopremovalareproficientatclassswitchrecombination,howeverBcells containunrepairedbreaksandchromosomefusionsatIgH.Recurrentoncogenictranslocations involvingIgHdistinguishmanyhumanlymphoidmalignancies.Thesetranslocationsoriginatefrom mis-repairedDNAdoublestandbreaks(DSBs)generatedduringnormallymphocytedevelopment. OurgoalistodeterminehowpersistentRloopsimpedeDNArepairduringCSR,andtheroleRloop metabolismplaysinsuppressinggenomeinstabilityatIgH.WehypothesizethatpersistentRloops blockefficientDNArepairbynon-homologousendjoiningattheimmunoglobulinheavychain locusduringclassswitchrecombination,leadingtopersistent,unrepairedbreaks.Totestthis hypothesis,twomousemodelswillbeemployed:theSETXmutantlackstheSenataxin(SETX) helicasethatunwindsRloops;?andRnaseh2bisdefectivefortheRNaseH2nucleasethatspecifically digeststheRNAcomponentofRloops(RNH2B).Wewillfunctionallydissecttheconsequencesof aberrantRloopformationonDNArepairandchromosomefusionsarisingduringCSRinSETX-/-, RNH2Bf/f,andSETX-/-RNH2Bf/fcells(Aim1).TodefinetheimpactpersistentRloopshaveonNHEJ, wewillcharacterizeDNArepairproteinrecruitmentinSETX-/-,RNH2Bf/f,andSETX-/-RNH2Bf/fcells (Aim2).WewillalsoidentifygenomiclociinvolvedinIgHtranslocationsusinghigh-throughput genome-widetranslocationsequencing(HTGTS-Seq),incollaborationwithDr.Feyredoun Hormozdiari.Finally,wewilldefinethemolecularpathwaysdrivingthefrequentchromosomefusions observedinSETX-/-RNH2Bf/fcells(Aim3).OurworkwilldefinehowpersistentRloopsinterferewith classswitchrecombination,leadingtounrepairedbreaks,andwilluncoverthemolecular mechanismspromotingchromosomefusionsatIgH.EnzymesregulatingRloopmetabolismwillalso provideanattractivetargetfordevelopingnovelcancertreatment.